JP7145051B2 - inverted microscope - Google Patents

Description

The present invention relates to an inverted microscope.

In recent years, a method of evaluating drug efficacy by acquiring microscopic image data of three-dimensional cultured cells such as spheroids and organoids and then screening the acquired microscopic image data using image analysis technology has attracted attention. In addition, in recent years, large investments have been made in medical care and drug discovery, and large-scale research and development of medical care and drug discovery is underway. In response to these investments and research and development, significant progress has been made in the experimental environment surrounding spheroids and organoids. One of them is the improvement of culture vessels.

Conventionally, culture vessels such as microplates having a spherical internal structure, that is, a spherical bottom structure existed. Even if it was possible to produce it, it was not able to withstand high-definition observation with a microscope. In fact, when conducting microscopic observation, researchers had no choice but to transfer samples such as prepared spheroids or organoids to a petri dish having a flat bottom before observation.

A pipette is often used to transfer samples, but transfer using a pipette can damage or contaminate the sample, and is time consuming because it requires careful handling. , there was a problem that it was costly. On the other hand, in recent years, microplates capable of improving this problem have been put on the market (see, for example, Non-Patent Document 1).

In the microplate described in Non-Patent Document 1, the bottom of the well is formed of a thin film, and the bottom has a convex surface with an outer spherical structure in the center. cause agglomeration. The microplate described in Patent Document 1 has a uniform bottom thickness because the bottom is molded with a film, and the optical performance is deteriorated compared to conventional microplates with non-uniform bottom thickness. The effect on the surface was small, and it was able to withstand microscopic observation. Researchers can use this microplate to prepare samples such as spheroids and organoids, and observe them under a microscope without having to transfer these samples to other containers for observation, improving work efficiency. did.

CORNING, "Microplate", [online], [searched in September 2018], Internet <URL: https://www.corning.com/jp/jp/products/life-sciences/products/surfaces/ultra- low-attachment-surface.html>

However, although the microplate described in Non-Patent Document 1 can reduce the deterioration of optical performance, it has not reached the point where the optical performance of the microscope can be maximized. , there was a need to reduce the optical influence of the bottom with a convex structure. In particular, if a dry objective lens is used as it is in the microplate described in Non-Patent Document 1, the medium inside the well and the bottom of the well act as a convex lens, resulting in significant deterioration in optical performance.

The present invention has been made in view of the above-mentioned circumstances, and uses a culture vessel having a structure that does not require the transfer of samples, thereby improving work efficiency and improving optical performance due to the shape of the bottom of the culture vessel. It is an object of the present invention to provide an inverted microscope capable of observing a sample with high definition by suppressing the deterioration of .

In order to achieve the above object, the present invention provides the following means.
A first aspect of the present invention is an inverted microscope for observing a sample contained together with a medium in a sample container having a bottom part at least partially optically transparent and having a convex lower surface, an objective lens arranged with its tip facing the lower surface of the bottom of the sample container; a sighting section for changing a relative position between the objective lens and the sample container in the optical axis direction of the objective lens; a movable stage that supports a sample container movably in a direction perpendicular to the optical axis; It has a transparent portion through which light from a sample can pass, and can hold a first liquid between the upper surface of the transparent portion and the lower surface of the bottom portion of the sample container in contact with the lower surface of the bottom portion. an optical member , an injection device that directly or indirectly supplies the first liquid between the upper surface of the optical member and the lower surface of the bottom of the sample container, and by controlling the injection device and driving the movable stage after filling the space between the lower surface of the bottom of the other sample container adjacent to the sample container arranged on the optical axis and the upper surface of the optical member with the first liquid. and a control device for moving another of the sample containers on the optical axis by moving the sample container, wherein a plurality of the sample containers are arranged in an array, and the optical member moves between at least two of the sample containers adjacent to each other. The inverted microscope has a size capable of holding the first liquid between the lower surface of each of the bottoms and the upper surface of the optical member .
A second aspect of the present invention is an inverted microscope for observing a sample housed together with a medium in a sample container having a bottom part at least partially optically transparent and having a convex lower surface, an objective lens arranged with its tip facing the lower surface of the bottom of the sample container; a sighting section for changing a relative position between the objective lens and the sample container in the optical axis direction of the objective lens; a movable stage that supports a sample container movably in a direction perpendicular to the optical axis; It has a transparent portion through which light from a sample can pass, and can hold a first liquid between the upper surface of the transparent portion and the lower surface of the bottom portion of the sample container in contact with the lower surface of the bottom portion. an optical member; and a supporting member capable of supporting the optical member while maintaining a constant relative position between the optical member and the objective lens, wherein the supporting member is integral with the optical member and The objective lens is an immersion objective lens, the optical member has at least one through-hole at a position out of the effective light flux of the immersion objective lens, and the optical member and an inverted microscope in which one space having the through hole as an opening is formed by the support member and the immersion objective lens.

According to the inverted microscope according to this aspect, the light emitted downward from the sample through the bottom of the sample container is collected by the objective lens after passing through the first liquid and the transparent portion of the optical member. be done. Therefore, the sample can be observed by detecting the light from the sample condensed by the objective lens while the positions of the objective lens and the sample container are adjusted by the sighting portion and the movable stage.

In this case, the optical member is formed between the upper surface of the transparent portion and the lower surface of the bottom of the sample container so that the first liquid can be held in contact with the lower surface of the bottom of the sample container. , By filling the first liquid between the lower surface of the bottom of the sample container and the upper surface of the transparent part of the optical member, the medium and the bottom of the sample container are more stable than when the lower surface of the bottom of the sample container is in contact with air. It is possible to reduce the difference in refractive index between and to suppress deterioration of optical performance due to the shape of the bottom of the sample container. In addition, since the relative position between the optical member and the objective lens is kept constant regardless of the movement of the sighting section, even if the observation position in the depth direction of the sample is changed, it is possible to prevent the spherical aberration from changing. can be done. As a result, it is possible to realize high-definition observation of the sample while improving work efficiency by using a culture vessel having a structure that does not require the transfer of the sample.

In the above aspect, the optical member may have an optical characteristic that corrects degradation of optical performance caused by the shape of the lower surface of the bottom of the sample container.
With this configuration, even when the medium, the bottom of the sample container, and the first liquid have different refractive indexes, the optical member corrects optical aberration caused by the shape of the lower surface of the bottom of the sample container, A sample can be observed with higher definition.

In the above aspect, the optical member may be a plane-parallel plate.
With this configuration, the manufacturing cost can be reduced by the simple shape of the optical member.

In the above aspect, the optical member may have a water-repellent treatment applied to at least a part of the upper surface of the transparent portion.
With this configuration, the first liquid held on the upper surface of the transparent portion of the optical member is easily collected in the form of droplets. Thereby, it is possible to easily secure the height of the first liquid on the upper surface of the transparent portion of the optical member.

In the above aspect, the first liquid may have the same refractive index as the medium.
With this configuration, the refractive index difference between the medium and the first liquid can be ignored, and optical aberrations caused by the refractive index difference between the medium, the bottom of the sample container, and the first liquid can be easily reduced.

In the above aspect, a support member capable of supporting the optical member while maintaining a constant relative position between the optical member and the objective lens is provided, the support member being integrated with the optical member, and It may be formed so as to be attachable to the objective lens.
With this configuration, if a plurality of pairs of optical members and support members are prepared according to the shape of the sample container, it is possible to easily change to an appropriate optical member according to the shape of the sample container to be used.

In the above aspect, the support member may be configured to be positionally adjustable in the optical axis direction.
With this configuration, the distance between the objective lens and the optical member can be adjusted according to the shape of the bottom of the sample container, the size or height of the sample, the viscosity of the first liquid, the wettability of the upper surface of the transparent portion of the optical member, and the like. Can be set freely.

In the above aspect, the objective lens is an immersion objective lens, and a second liquid, gel, or water-absorbing polymer is filled between the tip of the immersion objective lens and the lower surface of the transparent portion of the optical member. It is also possible to
With this configuration, a higher resolution image of the sample can be obtained compared to using a dry objective lens.

In the above aspect, the second liquid may be non-volatile.
With this configuration, when the second liquid is used, replenishment of the second liquid is unnecessary, and maintenance is facilitated.

In the above aspect, the refractive index of the first liquid and the refractive index of the second liquid may be the same.
This configuration allows negligible refractive index differences between the first and second liquids, reducing optical aberrations resulting from refractive index differences between the medium, the bottom of the sample vessel, the first liquid, and the second liquid. becomes easier.

In a second aspect, the objective lens is an immersion objective lens, the optical member has at least one through-hole at a position out of the effective light flux of the immersion objective lens, and the optical member and the support The member and the immersion objective lens form one space having the through hole as an opening . .

With this configuration, the space formed by the optical member, the support member, and the immersion objective lens is filled with the first liquid, and the first liquid seeps out from the through-hole. Liquid can be supplied.

In the above aspect, a plurality of the sample containers may be arranged in an array, and the distance between the optical axis and the through hole in the direction orthogonal to the optical axis may be substantially equal to the arrangement pitch of the sample containers.
With this configuration, the first liquid can be easily supplied between the lower surface of the bottom of another sample container adjacent to the sample container arranged on the optical axis of the objective lens and the upper surface of the optical member.

In the above aspect, the inverted microscope may include a liquid injection device that directly or indirectly supplies the first liquid between the upper surface of the optical member and the lower surface of the bottom of the sample container. good.
With this configuration, the first liquid can be easily filled between the upper surface of the transparent portion of the optical member and the lower surface of the bottom of the sample container by the liquid injection device, as compared with the case of manual liquid injection. .

In the above aspect, a plurality of the sample containers are arranged in an array, and the optical member is positioned between the lower surface of each of the bottoms of at least two adjacent sample containers and the upper surface of the optical member. may have a size capable of holding a liquid of
With this configuration, the first liquid can also be filled in advance between the lower surface of the bottom of another sample container adjacent to the sample container arranged on the optical axis of the objective lens and the upper surface of the optical member. can.

In the first aspect, the inverted microscope controls the liquid injection device so that the lower surface of the bottom of the other sample container adjacent to the sample container arranged on the optical axis and the optical member A control device is provided for moving the other sample container onto the optical axis by driving the movable stage after filling the space between the sample container and the upper surface with the first liquid .

With this configuration, when another sample container adjacent to the sample container arranged on the optical axis of the objective lens is moved onto the optical axis of the objective lens by the control device, the lower surface of the bottom of the other sample container and the lower surface of the other sample container The lower surface of the bottom of the other sample container and the upper surface of the transparent portion of the optical member moved on the optical axis of the objective lens by being dragged by the surface tension of the first liquid filled between the upper surface of the optical member and the lower surface of the bottom of the other sample container. The filled state of the first liquid is maintained even between and. This prevents the liquid from running out due to the movement of the sample container, and the first liquid already exists between the lower surface of the bottom of the sample container and the upper surface of the transparent portion of the optical member when the objective lens is moved onto the optical axis. By being filled, the target sample can be observed immediately, and the throughput can be improved.

In the above aspect, the control device controls the movable stage to cause the other sample container to pass once on the optical axis and then to return the other sample container to the optical axis. may be moved on the optical axis.
With this configuration, the first liquid can be evenly filled around the optical axis of the objective lens, and the reliability of the filling of the first liquid can be improved.

In the above aspect, the control device controls the liquid injection device so that, during observation of the sample in the sample container arranged on the optical axis, the bottom portion of the other adjacent sample container is observed. The first liquid may be supplied between the lower surface of and the upper surface of the transparent portion of the optical member.
With this configuration, it is possible to save the user's trouble, reduce the time loss between the timing of observing the sample and the timing of injecting the first liquid, and further improve the throughput.

In the above aspect, the direction in which the movable stage is moved is set in advance along the direction in which the plurality of sample containers are arranged, and the liquid injection device is arranged behind the moving direction of the movable stage. It may have a mouth.
With this configuration, the first liquid can be efficiently supplied from the injection port of the injection device while moving the sample container.

In the above aspect, a plurality of the sample containers are arranged two-dimensionally, and at least three liquid injection devices are arranged at equal intervals around the optical axis corresponding to the arrangement direction of the sample containers. It is good also as having the above said injection port.
With this configuration, the first liquid can be efficiently supplied from any inlet regardless of which direction the sample container is moved onto the optical axis of the objective lens.

In the above aspect, the control device may control the supply of the first liquid from the liquid inlet in accordance with the movement of the movable stage.
With this configuration, it is possible to save the user's trouble and improve work efficiency.

According to the present invention, the use of a culture vessel having a structure that does not require sample transfer improves work efficiency while suppressing deterioration in optical performance due to the shape of the bottom of the culture vessel, thereby increasing sample quality. It is effective in being able to observe finely.

1 is a schematic configuration diagram of an inverted microscope according to a first embodiment of the present invention; FIG. 1 is a schematic configuration diagram of an inverted microscope that employs an optical member consisting of a plane-parallel plate; FIG. 1 is a schematic configuration diagram of an inverted microscope that employs an immersion objective lens; FIG. FIG. 5 is a schematic configuration diagram of an inverted microscope according to a second embodiment of the present invention; FIG. 10 is a vertical cross-sectional view of an inverted microscope for explaining how water is injected between the bottom of a well to be observed next and an optical member; FIG. 4 is a longitudinal sectional view of an inverted microscope for explaining a state in which water is filled between the bottom of a well to be observed next and an optical member; FIG. 11 is a schematic configuration diagram of an inverted microscope according to a third embodiment of the present invention; FIG. 8 is a plan view showing an example of a microplate used in the inverted microscope of FIG. 7; FIG. 11 is a plan view showing an optical member of an inverted microscope according to a modification of the third embodiment of the invention; FIG. 10 is a longitudinal sectional view of the optical member of FIG. 9; FIG. 11 is a longitudinal sectional view showing a modification of the optical member of FIGS. 9 and 10; FIG. 11 is a schematic configuration diagram of an inverted microscope according to a fourth embodiment of the present invention;

[First embodiment]
An inverted microscope according to a first embodiment of the present invention will be described below with reference to the drawings.
The inverted microscope 1 according to the present embodiment, for example, as shown in FIG. An objective lens 7 of a dry system that emits light, an aiming unit 9 that changes the relative position between the objective lens 7 and the well 5 in the optical axis direction of the objective lens 7, and the well 5 in a direction orthogonal to the optical axis K of the objective lens 7. an electric stage (movable stage) 11 that is movably supported by the optical member 11; an optical member 13 arranged between the objective lens 7 and the well 5; and a support member 15 that supports the optical member 13.

The microplate 3 is made of polystyrene, for example. This microplate 3 has, for example, a plurality of wells 5 that are arranged at equal intervals vertically and horizontally. Each well 5 of the microplate 3 is optically transparent and has a transparent bottom 6 of uniform thickness.

The bottom 6 of the well 5 has an inner spherical top surface 6a and a convex bottom surface 6b located outside the well 5. As shown in FIG. Also, the bottom portion 6 of the well 5 has a refractive index of, for example, 1.59. The culture solution W1 has a refractive index of, for example, 1.33. In this microplate 3, since the bottoms 6 of the wells 5 have a uniform thickness, the sample S prepared in the wells 5 can be observed with the inverted microscope 1 without being transferred to another container for observation. be able to.

The objective lens 7 is arranged in a state in which the tip thereof faces the lower surface 6b of the bottom portion 6 of the well 5 in a vertically upward posture. Hereinafter, the direction of the optical axis of the objective lens 7 is defined as the Z direction, and the directions perpendicular to the optical axis K of the objective lens 7 and perpendicular to each other are defined as the X direction and the Y direction.

A liquid collecting member 17 for collecting excess liquid is attached to the objective lens 7 .
The liquid collecting member 17 is formed, for example, in the shape of a flange that expands radially outward from the outer peripheral surface of the objective lens 7 . The liquid collecting member 17 is provided with a drainage channel 17a for discharging the collected excess liquid. The liquid may be drained by connecting a pump to the drainage channel 17a, or the liquid may be collected by a drain tank (not shown) by allowing the liquid to naturally fall from the drainage channel 17a by gravity.

The electric stage 11 can be fixed above the objective lens 7 with the microplate 3 placed horizontally. Further, the electric stage 11 has a motor (not shown), and can electrically move the microplate 3 in the X and Y directions.

The optical member 13 is, for example, a plate-like member having a uniform thickness, and is configured integrally with the support member 15 by being adhered to the support member 15 . Further, the optical member 13 is made of a material through which the light from the sample S can pass, and the entirety functions as a transparent portion. This optical member 13 can hold water (first liquid) W2 between the upper surface 13a of the optical member 13 and the lower surface 6b of the bottom 6 of the well 5 in contact with the lower surface 6b of the bottom 6 of the well 5. is formed in

Also, the optical member 13 has a shape and a refractive index that compensate for deterioration in optical performance that occurs between the culture solution W1, the water W2, and the bottom portion 6 of the well 5. FIG. Specifically, the optical member 13 has a uniform thickness, and has a shape that is slightly warped in a direction in which the upper surface 13a becomes a convex spherical surface. The upper surface 13a of the optical member 13 is subjected to a water-repellent treatment, and retains the water W2 without flowing down even if the upper surface 13a has a convex spherical shape.

For example, water (first liquid) W2 can be injected from a liquid injection nozzle (liquid supply port) 19 between the upper surface 13a of the optical member 13 and the lower surface 6b of the bottom 6 of the well 5 . The liquid injection nozzle 19 is fixed to the liquid collection member 17 . This liquid injection nozzle 19 is connected to a pump (not shown), and the water W2 is supplied by the operation of the pump. Water W2 has a refractive index of, for example, 1.33.

The support member 15 is detachably provided at the tip of the objective lens 7 . This supporting member 15 arranges the optical member 13 between the tip of the objective lens 7 and the bottom portion 6 of the well 5, and allows the relative positions of the optical member 13 and the objective lens 7 to be adjusted regardless of the operation of the sighting portion 9. The optical member 13 is supported while being kept constant.

The operation of the inverted microscope 1 having the above configuration will be described.
In order to observe the sample S contained in the well 5 of the microplate 3 with the inverted microscope 1 according to the present embodiment, the electric stage 11 is used to move the center position of the well 5 containing the sample S to be observed. It is arranged on the optical axis K of the objective lens 7 .

Next, a pump (not shown) is used to inject water W2 from the liquid injection nozzle 19 between the upper surface 13a of the optical member 13 and the lower surface 6b of the bottom portion 6 of the well 5. Water W2 is filled between 6 and the lower surface 6b. Then, the relative position of the objective lens 7 and the well 5 in the Z direction is adjusted by the aiming unit 9 .

In this state, when the sample S is irradiated with illumination light from a light source (not shown), the light emitted downward from the sample S through the bottom portion 6 of the well 5 passes through the water W2 and the optical member 13 and then reaches the objective. The light is collected by lens 7 . Therefore, the sample S can be observed by detecting the light from the sample S condensed by the objective lens 7 with a detector such as a photomultiplier tube or an imaging device such as a CCD.

In this case, by filling water W2 between the upper surface 13a of the optical member 13 and the lower surface 6b of the bottom 6 of the well 5, the lower surface 6b of the bottom 6 of the well 5 is in contact with the air. The difference in refractive index between the culture solution W1 and the bottom portion 6 of the well 5 can be made smaller than in the case where the well 5 has a bottom portion 6, thereby suppressing deterioration in optical performance due to the shape of the bottom portion 6 of the well 5.

Further, since the relative position between the optical member 13 and the objective lens 7 is kept constant by the support member 15 regardless of the operation of the aiming unit 9, even if the observation position in the depth direction of the sample S is changed, It is possible to prevent spherical aberration from changing.

Therefore, according to the inverted microscope 1 according to the present embodiment, the optical member 13 and the well 5 can be separated from the optical member 13 and the well 5 while improving work efficiency by using the well 5 having a structure that does not require the sample S to be transferred to another container during observation. High-definition observation of the sample S can be realized only by filling the space between the bottom portion 6 of the sample S and the water W2.

Further, since the optical member 13 is formed integrally with the support member 15 and detachable from the objective lens 7, a plurality of optical members 13 may be prepared according to the shape of the well 5 of the microplate 3. , can be easily changed to an appropriate optical member 13 according to the microplate 3 to be used.

In addition, since the upper surface 13a of the optical member 13 is subjected to a water-repellent treatment, the water W2 held on the upper surface 13a is easily formed into droplets. can be easily ensured. Further, by adopting the water W2 having the same refractive index as the culture solution as the first liquid, the difference in refractive index between the culture solution W1 and the water W2 can be ignored, and the culture solution W1 and the bottom of the well 5 can be disregarded. 6 and water W2 can reduce the optical aberration caused by the difference in refractive index.

In the present embodiment, the lower surface 6b of the bottom 6 of the well 5 of the microplate 3 is formed in a convex spherical shape, but the lower surface 6b of the bottom 6 may have a parabolic surface. In this case, the optical member 13 may have a shape and a refractive index that compensate for deterioration in optical performance caused by the shape of the lower surface 6b of the bottom 6 of the well 5 and the refractive index of the bottom 6. FIG.

It is only necessary to correct the deterioration of the optical performance that occurs according to the shape of the bottom 6 of the well 5. etc. may be adopted. That is, the optical member 13 possesses optical characteristics for correcting aberrations (optical characteristics) caused by the shape of the bottom portion 6 of the well 5, and the liquid filled between the bottom portion 6 of the well 5 and the optical member 13. Any material may be used as long as it has a shape and surface characteristics that can be retained.

In the present embodiment, the upper surface 13a of the optical member 13 has a spherical (curved) shape in order to correct aberrations strictly. It is good also as adopting parallel flat plates, such as.

In this case, for example, as shown in FIG. 2, a transparent optical member 21 made of a plane-parallel plate is inserted between the tip of the objective lens 7 and the bottom 6 of the well 5, and the lower surface 6b of the bottom 6 of the well 5 and Water W2 may be filled between the upper surface 21a of the optical member 21 and the upper surface 21a. With this configuration, the lens effect caused by the bottom portion 6 of the well 5 and the culture solution W1 in the well 5 can be substantially canceled, and deterioration of optical performance can be largely avoided.

Also, in this embodiment, the dry objective lens 7 is used, but instead of this, for example, a liquid immersion objective lens 23 may be used as shown in FIG. In this case, for example, silicone liquid (second liquid) W3 may be filled between the tip lens 23a of the immersion objective lens 23 and the optical member 21 .

With this configuration, a higher resolution image of the sample S can be obtained than when the dry objective lens 7 is used. In this modified example, a gel or a water-absorbing polymer may be used instead of the silicone liquid W3. FIG. 3 shows the configuration employing the optical member 21, but as described above, the optical member 13 whose shape and refractive index are set according to the optical characteristics of the bottom portion 6 of the well 5 is used. It may be adopted.

[Second embodiment]
Next, an inverted microscope according to a second embodiment of the invention will be described.
As shown in FIG. 4, the inverted microscope 1 according to the present embodiment employs an optical member 21 made of plane-parallel glass in place of the optical member 13, and further includes a liquid injection device 25 for supplying water W2 and an injection device 25 for supplying water W2. This embodiment differs from the first embodiment in that a control device 27 for controlling the liquid device 25 is provided.
In the following, parts having the same configuration as the inverted microscope 1 according to the first embodiment are denoted by the same reference numerals, and descriptions thereof are omitted.

In this embodiment, the optical member 21 has an oval shape and is adhered to the tip of the objective lens 7 . This optical member 21 has a size capable of holding water W2 between the lower surface 6b of each bottom 6 of at least two adjacent wells 5 and the upper surface 21a of the optical member 21. As shown in FIG. Further, the upper surface 21a of the optical member 21 is subjected to a water-repellent treatment.

The liquid injection device 25 includes a liquid injection nozzle 19 that injects liquid between the upper surface 21 a of the optical member 21 and the lower surface 6 b of the bottom portion 6 of the well 5 , and a pump 20 that sends water W 2 in a tank (not shown) to the liquid injection nozzle 19 . and The liquid injection nozzle 19 is fixed to the liquid collecting member 17, and the lower surface 6b of the bottom portion 6 of another well 5 adjacent to the well 5 arranged on the optical axis K of the objective lens 7 and the upper surface 21a of the optical member 21. and is arranged so that water W2 can be injected between them.

The control device 27 includes, for example, a CPU (Central Processing Unit), a main storage unit such as a ROM (Read Only Memory) and a RAM (Random Access Memory), an auxiliary storage unit such as a HDD (Hard Disk Drive), and data. An output unit for output and an external interface for exchanging various data with an external device (none of them are shown) are provided. Various programs are stored in the auxiliary storage unit, and the CPU reads the programs from the auxiliary storage unit to the main storage unit such as RAM, and executes the programs to control the motorized stage 11 and the injection device 25. .

For example, the control device 27 controls the pump 20 of the liquid injection device 25 by executing the liquid injection program so that the water W2 is placed at a position separated from the optical axis K of the objective lens 7 by one well pitch of the microplate 3 . is injected. Further, by executing the driving program, the control device 27 controls the lower surface 6b of the bottom portion 6 of the well 5 and the optical member 21, which are arranged at a position separated by one well pitch of the microplate 3 from the optical axis K of the objective lens 7. The well 5 is moved onto the optical axis K of the objective lens 7 by driving the motorized stage 11 in a state in which water W2 is filled between the well 5 and the upper surface 21a.

The operation of the inverted microscope 1 having the above configuration will be described.
In order to observe the sample S contained in the well 5 of the microplate 3 with the inverted microscope 1 according to this embodiment, first, the motorized stage 11 is driven by the control device 27, so that as shown in FIG. First, the well 5 to be observed is stopped at a position separated from the optical axis K of the objective lens 7 by one well pitch of the microplate 3 .

Next, by driving the pump 20 of the liquid injection device 25 by the control device 27, liquid is injected from the liquid injection nozzle 19, and the liquid is injected at a position away from the optical axis K of the objective lens 7 by one well pitch of the microplate 3. , the space between the lower surface 6b of the bottom 6 of the well 5 to be observed and the upper surface 21a of the optical member 21 is filled with water W2.

Next, by driving the motorized stage 11 by the control device 27, the microplate 3 is moved by one well pitch as shown in FIG. placed above. At this time, the water W2 filled between the lower surface 6b of the bottom 6 of the well 5 to be observed and the upper surface 21a of the optical member 21 moves along the optical axis K of the objective lens 7 due to surface tension as the microplate 3 moves. The filled state of the water W2 is maintained by being dragged up.

In this state, after adjusting the relative position of the objective lens 7 and the well 5 in the Z direction by the aiming unit 9, the sample S housed in the well 5 arranged on the optical axis K of the objective lens 7 is observed. is started, the pump 20 of the injection device 25 is driven by the control device 27, and as shown in FIG. Liquid is injected between the lower surface 6b of the bottom portion 6 of the well 5 and the upper surface 21a of the optical member 21 . The excessively supplied water W2 is, for example, collected by a liquid collection member 17 as shown in FIG. 6 and discharged from a drainage channel 17a. The above steps are sequentially performed for each well 5 .

Here, if the lower surface 6b of the bottom 6 of the microplate 3 has a flat shape over the entire area, the samples S in the plurality of wells 5 can be observed by one injection of liquid. If the shape of 6b is not flat, when the microplate 3 is moved, the water W2 filled in the lower surface 6b of the bottom 6 of the well 5 is interrupted by the step of the bottom 6 of the well 5, resulting in liquid depletion. . Therefore, it is necessary to inject liquid into each well 5 to be observed. Injecting the liquid between the optical member 21 takes time and the throughput cannot be increased.

In contrast, according to the inverted microscope 1 according to the present embodiment, when the well 5 to be observed is moved onto the optical axis K of the objective lens 7, the lower surface 6b of the bottom 6 of the well 5 and the optical member 21. Since water W2 has already been filled between the well 5 and the upper surface 21a of the objective lens 7, the sample S in the well 5 arranged on the optical axis K of the objective lens 7 can be immediately observed and photographed. Therefore, the control of the motorized stage 11 and the injection device 25 by the control device 27 saves the user's trouble, reduces the time loss between the timing of observing the sample S and the timing of injecting the water W2, and improves the throughput. can do.

In this embodiment, injection may continue during the movement of the microplate 3 to the next well 5 .
With this configuration, it is possible to prevent the liquid amount from decreasing due to the water W2 being dragged along with the movement of the microplate 3, so that the water W2 can be stably filled.

When arranging the well 5 to be observed next on the optical axis K of the objective lens 7, the well 5 to be observed next is caused to pass through the optical axis K of the objective lens 7 once, and then again on the optical axis K. You can also put it back up.
With this configuration, the water W2 can be more evenly filled around the optical axis K of the immersion objective lens 23, and the reliability of the filling of the water W2 can be improved.

In this embodiment, polystyrene is used as the member forming the bottom portion 6 of the well 5. While the refractive index of water is 1.33, the refractive index of polystyrene is considerably higher than that of water. It is about 1.59. Therefore, optical aberration occurs due to the refractive index difference between the bottom portion 6 of the well 5 made of polystyrene and water. Therefore, ideally, it is desirable to use a material having a refractive index close to that of water, such as a fluororesin having a refractive index of 1.34, as the member forming the bottom portion 6 of the well 5 . Also, the thinner the bottom portion 6 of the well 5, the better.

[Third embodiment]
Next, an inverted microscope according to a third embodiment of the invention will be described.
For example, as shown in FIG. 7, the inverted microscope 1 according to the present embodiment employs an immersion objective lens 23 instead of the objective lens 7, and the control device 27 drives the electric stage 11 to move the microplate 3 zigzag in the X and Y directions, which is different from the first and second embodiments.
In the following description, the same reference numerals are given to the portions having the same configuration as the inverted microscope 1 according to the first and second embodiments, and the description thereof will be omitted.

The microplate 3 has, for example, 24 wells 5 arranged in 4 rows and 6 columns, as shown in FIG. In FIG. 8, the wells 5 arranged in the first row are A1 to A6, the wells 5 arranged in the second row are B1 to B6, the wells 5 arranged in the third row are C1 to C6, and 4 The wells 5 arranged in the row are D1 to D6.

In this embodiment, as shown in FIG. 8, the optical member 21 has a disk shape. This optical member 21 is fixed to the liquid collecting member 17 and arranged at a small distance from the tip lens 23 a of the immersion objective lens 23 .

As shown in FIGS. 7 and 8, the liquid injection device 25 has three liquid injection nozzles 19A and 19B arranged at equal intervals around the optical axis K of the immersion objective lens 23 corresponding to the array direction of the wells 5. , 19C. In order to observe the samples S in all the wells 5 in the microplate 3, it is necessary to sequentially move the motorized stage 11 in three directions. One direction is required for return, but observation and imaging are not performed during return. The three liquid injection nozzles 19A, 19B, and 19C are arranged, for example, at three locations indicated by ▴ in FIG.

In the program of the controller 27, the direction in which the electric stage 11 is moved along the array direction of the plurality of wells 5 is set in advance. The control device 27 selectively controls the liquid injection nozzle 19 that injects liquid from among the three liquid injection nozzles 19 in accordance with the moving direction of the electric stage 11 .

A space between the lower surface 23b of the optical member 21 and the tip lens 23a of the immersion objective lens 23 is preferably filled with a non-volatile liquid such as silicone liquid W3. With this configuration, there is an advantage that a commercially available objective lens can be used as it is without modification such as bonding the optical member 13 to the tip of the immersion objective lens 23 as in the second embodiment. In addition, since the silicone liquid W3 is non-volatile, maintenance is easy without replenishment of the silicone liquid W3.

In this embodiment, the sample S is, for example, three-dimensionally cultured cells such as spheroids, and the refractive index of the sample S is close to that of water. The sample S is immersed in the culture solution W1, and the space between the upper surface 21a of the optical member 21 and the lower surface 6b of the bottom 6 of the well 5 is filled with water W2. There is no refractive index difference between the bottom 6 of and the water W2. Therefore, even if the observation position of the sample S is changed in the Z direction, the total liquid thickness does not change, and the amount of spherical aberration does not change regardless of the observation position of the sample S. As a result, by adjusting the correction ring (not shown) of the immersion objective lens 23 only once, it is possible to greatly reduce the deterioration of the optical performance inherent in the optical system. The liquid thickness of the silicone liquid W3 does not change regardless of the observation position of the sample S in the Z direction. Therefore, no change occurs in spherical aberration.

The operation of the inverted microscope 1 having the above configuration will be described.
In order to observe the sample S contained in the wells 5 of the microplate 3 with the inverted microscope 1 according to this embodiment, the electric stage 11 is driven by the control device 27 to move the microplate 3 in a zigzag manner. At the same time, the pump 20 of the injection device 25 is driven by the control device 27, so that the next observation object arranged at a position separated from the optical axis K of the objective lens 7 by one well pitch of the microplate 3 Water W2 is injected from one of the injection nozzles 19 between the lower surface 6b of the bottom portion 6 of the well 5 and the upper surface 21a of the optical member 21. As shown in FIG.

Specifically, first, the well 5 of A1 to be observed first is moved to a position away from the optical axis K of the liquid immersion objective lens 23 by one well pitch of the microplate 3, and the liquid injection nozzle 19A to A1 is moved. Water W2 is filled between the lower surface 6b of the bottom portion 6 of the well 5 and the upper surface 21a of the optical member 21. As shown in FIG.

Next, by moving the microplate 3 by one well pitch, the well 5 of A1 is placed on the optical axis K of the immersion objective lens 23 together with the water W2. Then, while the sample S in the well 5 of A1 is observed, the solution is injected between the bottom surface 6b of the bottom 6 of the well 5 of A2 to be observed next adjacent to the well 5 of A1 and the upper surface 21a of the optical member 21. Water W2 is filled from the nozzle 19A.

Next, the wells A2 to A5 are sequentially moved onto the optical axis K of the immersion objective lens 23 together with the water W2, and the samples S in the wells A2 to A5 are sequentially observed. Between the lower surface 6b of the bottom portion 6 of the well 5 and the upper surface 21a of the optical member 21 from A3 to A6 adjacent to the well 5, water W2 is sequentially filled from the liquid injection nozzle 19A.

Next, the well 5 of A6 is moved onto the optical axis K of the immersion objective lens 23 together with the water W2, and the sample S of the well 5 of A6 is observed, and the lower surface 6b of the bottom 6 of the well 5 of B6 and the optical member are observed. Water W2 is filled from the liquid injection nozzle 19B between the upper surface 21a of 21 and the upper surface 21a.
Next, the well 5 of B6 is moved onto the optical axis K of the liquid immersion objective lens 23 together with the water W2, the sample S of the well 5 of B6 is observed, and the lower surface 6b of the bottom 6 of the well 5 of B5 and the optical member are observed. Water W2 is filled between the upper surface 21a of 21 and the liquid injection nozzle 19C.

Next, the wells 5 from B5 to B2 are sequentially moved onto the optical axis K of the immersion objective lens 23 together with the water W2, and the samples S in the wells 5 from B5 to B2 are sequentially observed. Water W2 is filled in order from the liquid injection nozzle 19C between the lower surface 6b of the bottom 6 of the well 5 and the upper surface 21a of the optical member 21 from B4 to B1 adjacent to the well 5 of .

Next, the well 5 of B1 is moved onto the optical axis K of the immersion objective lens 23 together with the water W2, the sample S of the well 5 of B1 is observed, and the lower surface 6b of the bottom 6 of the well 5 of C1 and the optical member are observed. Water W2 is filled from the liquid injection nozzle 19B between the upper surface 21a of 21 and the upper surface 21a.
Next, the well 5 of C1 is moved onto the optical axis K of the immersion objective lens 23 together with the water W2, the sample S of the well 5 of C1 is observed, and the lower surface 6b of the bottom 6 of the well 5 of C2 and the optical member are observed. 21 is filled with water W2 from the liquid injection nozzle 19A.

Next, the wells 5 from C2 to C5 are sequentially moved onto the optical axis K of the liquid immersion objective lens 23 together with the water W2, and the samples S in the wells 5 from C2 to C5 are sequentially observed. Between the lower surface 6b of the bottom portion 6 of the well 5 and the upper surface 21a of the optical member 21 from C3 to C6 adjacent to the well 5 of C3 to C6, water W2 is sequentially filled from the liquid injection nozzle 19A.

Next, the well 5 of C6 is moved onto the optical axis K of the liquid immersion objective lens 23 together with the water W2, the sample S of the well 5 of C6 is observed, and the lower surface 6b of the bottom 6 of the well 5 of D6 and the optical member are observed. Water W2 is filled from the liquid injection nozzle 19B between the upper surface 21a of 21 and the upper surface 21a.
Next, the well 5 of D6 is moved along with the water W2 onto the optical axis K of the immersion objective lens 23, the sample S of the well 5 of D6 is observed, and the lower surface 6b of the bottom 6 of the well 5 of D5 and the optical member are observed. Water W2 is filled between the upper surface 21a of 21 and the liquid injection nozzle 19C.

Next, the wells 5 from D5 to D2 are sequentially moved onto the optical axis K of the immersion objective lens 23 together with the water W2, and the samples S in the wells D5 to D2 are sequentially observed. Between the lower surface 6b of the bottom portion 6 of the well 5 and the upper surface 21a of the optical member 21 from D4 to D1 adjacent to the well 5, water W2 is sequentially filled from the liquid injection nozzle 19C.
Finally, the well 5 of D1 is moved onto the optical axis K of the immersion objective lens 23 together with the water W2, and the sample S in the well 5 of D1 is observed.

As described above, according to the inverted microscope 1 according to the present embodiment, by moving the microplate 3 zigzag in the X and Y directions to minimize the moving distance of the electric stage 11, each well can be 5 sample S can be observed with high efficiency. Further, by injecting the liquid from the liquid injection nozzle 19 arranged behind the electric stage 11 in the moving direction, the water W2 can be efficiently supplied while changing the well 5 to be observed.

In general, the material forming the bottom portion 6 of the well 5 is polypropylene, polystyrene, or fluorine-based resin, and their refractive indices are 1.34 to 1.59. Therefore, in this embodiment, it is desirable that the material forming the well 5 is a fluorine-based resin (refractive index 1.34) having a refractive index close to that of water. Furthermore, it is desirable that the thickness of the bottom portion 6 of the well 5 is thin.

In this embodiment, the immersion objective lens 23 is used, but a dry objective lens 7 may be used. If a dry objective lens 7 is used, the effect of improving the optical performance is even greater. When the sample S is observed through the bottom portion 6 of the well 5 having a convex lower surface 6b using the dry objective lens 7, the bottom portion 6 of the well 5 and the culture medium W1 in the well 5 can be seen as they are. However, by filling the gap between the optical member 21 and the bottom 6 of the well 5 with water W2, the refractive index difference can be reduced and the well The degradation of optical performance caused by the bottom portion 6 of the well 5 and the culture medium W1 in the well 5 can be greatly reduced.

If the refractive index of the culture medium W1 in the well 5, the refractive index of the bottom 6 of the well 5, and the refractive index of the water W2 can be made equal, the objective lens 7 can be adjusted by adjusting the correction ring for correcting spherical aberration once. The optical performance can be exhibited without deteriorating at all just by carrying out.

For example, the culture medium W1 in the well 5 is a clearing solution: Scale (refractive index 1.49), the material of the bottom 6 of the well 5 is polypropylene (refractive index 1.49), the optical member 13 and the bottom of the well 5 6 is the same clearing solution as the culture medium W1: Scale (refractive index: 1.49), the bottom 6 of the well 5 does not affect the optical system. Further, if the optical member 21 made of parallel plane glass is adopted instead of the optical member 13, the original optical performance of the objective lens 7 can be maximized without deterioration by adjusting the correction ring (not shown) only once. can do.

In this embodiment, the substance filled between the lower surface 21b of the optical member 21 and the tip lens 23a of the immersion objective lens 23 may be a gel substance or a water-absorbing polymer. . The liquid used for these gel-like substances and polymers may be a non-volatile liquid.

Further, in the present embodiment, for example, as shown in FIGS. 9 and 10, the optical member 21 has a disk shape and has an edge 21c rising from the upper surface 21a in the thickness direction over the entire circumferential direction. It is also possible to have In the examples shown in FIGS. 9 and 10, the optical member 21 has a size that allows liquid to be filled between five adjacent wells 5 of the microplate 3 . Since the optical member 21 has the edge 21c, the space between the optical member 21 and the bottom portion 6 of the well 5 can be stably filled with the water W2.

Further, the optical member 13 having a shape and a refractive index that correct deterioration of optical performance occurring at the bottom portion 6 of the well 5 may have an edge 13c rising from the upper surface 13a in the thickness direction over the entire circumferential direction. . For example, in the example shown in FIG. 11, the optical member 13 has a uniform thickness, a shape slightly curved in the direction in which the upper surface 13a becomes a convex spherical surface, and a It has an edge 13c.

[Fourth embodiment]
Next, an inverted microscope according to a fourth embodiment of the invention will be described.
As shown in FIG. 12, the inverted microscope 1 according to the present embodiment is the first to third optical members in that the optical member 21 has at least one through-hole 21d at a position out of the effective light beam of the immersion objective lens 23. Different from the embodiment.
In the following, parts having the same configuration as the inverted microscope 1 according to the first to third embodiments are denoted by the same reference numerals, and descriptions thereof are omitted.

In this embodiment, the optical member 21 is a plane-parallel glass, is fixed to the liquid collection member 17 functioning as a support member, and is spaced apart from the tip lens 23 a of the immersion objective lens 23 . This optical member 21 has one small through hole 21d at a position separated from the optical axis K of the liquid immersion objective lens 23 by one pitch of the wells 5 of the microplate 3 . The optical member 21, liquid collecting member 17, and immersion objective lens 23 form one space 29 which can be filled with water W2 and whose opening is the through hole 21d.

Further, in this embodiment, the liquid collecting member 17 is provided with an injection port 17b, and the liquid can be injected into the space 29 from the injection port 17b by a pump (not shown).
An O-ring 31 is provided between the liquid collecting member 17 and the immersion objective lens 23 . The O-ring 31 allows the liquid collecting member 17 to move in the optical axis direction of the immersion objective lens 23 , thereby changing the distance between the immersion objective lens 23 and the optical member 21 . The O-ring 31 also functions as a liquid shield.

According to the inverted microscope 1 according to this embodiment, the space 29 formed by the optical member 13, the liquid collecting member 17, and the liquid immersion objective lens 23 is filled with water W2, and the water W2 seeps out from the through hole 21d. Thus, the water W2 can be supplied to the upper surface 21a of the optical member 21. As shown in FIG.

Further, unlike the case where the optical member 21 is adhered to the tip lens 23a of the immersion objective lens 23, the optical member 21 can be moved in the optical axis direction of the immersion objective lens 23, so that the shape of the bottom portion 6 of the well 5 can be changed. , the size (height) of the sample S, the viscosity of the first liquid used, the wettability of the upper surface of the optical member 21, etc., the distance between the immersion objective lens 23 and the optical member 21 can be freely set. .

Although the optical member 21 has one through hole 21 d in this embodiment, the optical member 21 may have a plurality of through holes 21 d in accordance with the observation route of the microplate 3 . In this case, the space 29 may be divided into a plurality of sections, and the water W2 may be supplied to the upper surface 21a of the optical member 21 from the through-holes 21d corresponding to the respective spaces by injecting liquid into each of the divided spaces. good. By controlling the injection, the water W2 is not wasted.

The inverted microscope 1 according to this embodiment can be realized by the objective lens 7 having a large WD.
For example, assume that the immersion objective lens 23 has a large NA and a long working distance of WD=8 mm. Although it is difficult to fill the space between the immersion objective lens 23 having a large WD and the lower surface 6b of the bottom portion 6 of the well 5 with the first liquid, 6b, the optical member 21 can stably fill the first liquid. Further, by making the position of the optical member 21 adjustable in the Z direction, it is possible to widely adapt to microplates 3 having different shapes of the bottoms 6 of the wells 5 .

In this embodiment, the pump 20 of the injection device 25 may be connected to the injection port 17b to supply the water W2. Further, as in the second and third embodiments, by driving the electric stage 11 and the pump 20 by the control device 27, the microplate 3 is moved and the top surfaces 13a and 21a of the optical members 13 and 21 and the bottom portions 6 of the wells 5 are moved. It is also possible to control the supply of water W2 between the lower surface 6b of the .

Although the embodiment of the present invention has been described in detail above with reference to the drawings, the specific configuration is not limited to this embodiment, and design changes and the like are included within the scope of the present invention. For example, the present invention is not limited to those applied to the above embodiments and modifications, but may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is not particularly limited. .

Further, for example, in each of the above embodiments, the optical members 13 and 21 are made of a transparent material, but the optical members 13 and 21 are mostly made of an opaque material, It is also possible to partially have a transparent portion through which light from S can pass.

1 inverted microscope 5 wells (sample container)
6 bottom portion 6b lower surface 7 objective lens 9 sighting portion 11 electric stage (movable stage)
13, 21 Optical member 15 Support member 19, 19A, 19B, 19C Injection nozzle (injection port)
21d through-hole 23 immersion objective lens 25 injection device 27 control device 29 space S sample W1 culture solution (medium)
W2 water (first liquid)
W3 silicone liquid (second liquid)

Claims (19)

An inverted microscope for observing a sample contained together with a medium in a sample container having a bottom part at least partially optically transparent and having a convex lower surface,
an objective lens arranged with its tip facing the lower surface of the bottom of the sample container;
an aiming unit that changes the relative position between the objective lens and the sample container in the direction of the optical axis of the objective lens;
a movable stage that supports the sample container so as to be movable in a direction orthogonal to the optical axis;
a transparent portion disposed between the objective lens and the sample container in a state in which the relative position with respect to the objective lens is maintained constant, and through which light from the sample can pass; an optical member capable of holding a first liquid in contact with the lower surface of the bottom of the sample container ;
a liquid injection device that directly or indirectly supplies the first liquid between the upper surface of the optical member and the lower surface of the bottom of the sample container;
By controlling the liquid injection device, the first liquid is introduced between the lower surface of the bottom of the other sample container adjacent to the sample container arranged on the optical axis and the upper surface of the optical member. a control device for moving the other sample container onto the optical axis by driving the movable stage after filling ;
A plurality of the sample containers are arranged in an array,
The optical member has a size capable of holding the first liquid between the lower surface of each of the bottoms of at least two adjacent sample containers and the upper surface of the optical member . The control device controls the movable stage to cause the other sample container to pass through the optical axis once and then return it to the optical axis, thereby moving the other sample container to the optical axis. 2. The inverted microscope according to claim 1 , wherein the microscope is moved to the The control device controls the liquid injection device so that during observation of the sample in the sample container arranged on the optical axis, the lower surface of the bottom of the other adjacent sample container and the optical 3. An inverted microscope according to claim 1 , wherein said first liquid is supplied between said member and the upper surface of said transparent portion of said member. a direction in which the movable stage is moved along the direction in which the plurality of sample containers are arranged is set in advance;
4. The inverted microscope according to any one of claims 1 to 3 , wherein said liquid injection device has a liquid injection port arranged behind in the moving direction of said movable stage. A plurality of the sample containers are arranged two-dimensionally,
5. The inverted microscope according to claim 4 , wherein said liquid injection device has at least three or more said liquid injection ports arranged at equal intervals around said optical axis corresponding to the arrangement direction of said sample containers. 6. An inverted microscope according to claim 4 , wherein said control device controls supply of said first liquid from said liquid inlet in accordance with movement of said movable stage. the objective lens is an immersion objective lens,
7. The inverted type according to any one of claims 1 to 6 , wherein a second liquid, gel, or water-absorbing polymer is filled between the tip of the immersion objective lens and the lower surface of the transparent portion of the optical member. microscope. 8. An inverted microscope according to claim 7 , wherein said second liquid is non-volatile. 9. The inverted microscope according to claim 7 , wherein the refractive index of said first liquid and the refractive index of said second liquid are the same . a support member capable of supporting the optical member while maintaining a constant relative position between the optical member and the objective lens;
10. The inverted microscope according to any one of claims 1 to 9 , wherein the supporting member is formed integrally with the optical member and attachable to the objective lens. An inverted microscope for observing a sample contained together with a medium in a sample container having a bottom part at least partially optically transparent and having a convex lower surface,
an objective lens arranged with its tip facing the lower surface of the bottom of the sample container;
an aiming unit that changes the relative position between the objective lens and the sample container in the direction of the optical axis of the objective lens;
a movable stage that supports the sample container so as to be movable in a direction orthogonal to the optical axis;
a transparent portion disposed between the objective lens and the sample container in a state in which the relative position with respect to the objective lens is maintained constant, and through which light from the sample can pass; an optical member capable of holding a first liquid in contact with the lower surface of the bottom of the sample container ;
a support member capable of supporting the optical member while maintaining a constant relative position between the optical member and the objective lens;
the support member is formed integrally with the optical member and attachable to the objective lens;
the objective lens is an immersion objective lens,
the optical member has at least one through-hole at a position out of the effective light flux of the immersion objective lens;
An inverted microscope in which the optical member, the support member, and the immersion objective lens form one space having the through hole as an opening . A plurality of the sample containers are arranged in an array,
12. The inverted microscope according to claim 11, wherein the distance between said optical axis and said through-hole in a direction perpendicular to said optical axis is substantially equal to the arrangement pitch of said sample containers. 13. The inverted microscope according to claim 11 , further comprising a liquid injection device that directly or indirectly supplies the first liquid between the upper surface of the optical member and the lower surface of the bottom of the sample container. . A plurality of the sample containers are arranged in an array,
14. The inverted apparatus according to claim 13, wherein the optical member has a size capable of holding the first liquid between the lower surface of each of the bottoms of at least two adjacent sample containers and the upper surface of the optical member. type microscope. 15. The inverted microscope according to any one of claims 10 to 14 , wherein said support member is configured to be positionally adjustable in said optical axis direction. 16. The inverted microscope according to any one of claims 1 to 15 , wherein the optical member has an optical characteristic that corrects degradation of optical performance caused by the shape of the lower surface of the bottom of the sample container. 16. An inverted microscope according to any one of claims 1 to 15, wherein said optical member is a plane-parallel plate. 18. The inverted microscope according to any one of claims 1 to 17 , wherein the optical member has a water-repellent treatment applied to at least a part of the upper surface of the transparent portion. 19. An inverted microscope according to any one of claims 1 to 18 , wherein said first liquid has a refractive index equivalent to that of said medium.

Images